Oligosaccharides and polysaccharides, as components of glycoproteins and glycolipids, are ubiquitous on cell surfaces where they function as cell-surface markers that protein receptors can recognize. Although such carbohydrate-mediated cell-cell interactions are of importance in many biological events, our understanding of the structure-function relationships has been very slow to develop because of the difficulty of synthesizing well-defined oligosaccharides for study.
Carbohydrate ligands play central roles in a wide variety of normal and abnormal biological recognition processes. Among their less benign roles, carbohydrates on cell surfaces have been implicated in chronic inflammation, in viral and bacterial infection, and in tumorigenesis and metastasis. Strategies to block the interactions between cell surface carbohydrates and their protein receptors could provide an effective means of preventing or treating various diseases. Antibodies directed to either the carbohydrate ligands or to their respective receptors represent one approach; another strategy is to deploy ligands which bind to the protein targets better than the natural cell surface carbohydrates. Consequently, there is great potential for, and a great amount of research into, the use of synthetic oligosaccharides as therapeutic agents.
One application of synthetic oligosaccharides is for the inhibition of cell adhesion. For example, a critical step in the inflammatory response is the adhesion of circulating neutrophils to the endothelial lining of blood vessels, which is an essential precursor to neutrophil infiltration of the surrounding tissue. The neutrophils initially adhere via carbohydrate ligands to adhesion molecules (selecting) and then, in the presence of pro-inflammatory chemokines, they bind more tightly via a family of cell adhesion molecules (CAMs) called integrins. The integrins incorporate carbohydrate ligands that bind to intracellular adhesion molecules (ICAMs) on the epithelium. There is currently great interest in synthetic carbohydrate ligands that might interfere with selectin and/or integrin binding; such agents are expected to be of use in the treatment of asthma, ARDS, reperfusion injury, multiple sclerosis, and various other chronic inflammatory diseases.
Another example of this application of synthetic carbohydrates is the inhibition of bacterial adhesion to human tissue. Many pathogenic bacteria adhere to carbohydrate ligands on the host cells, and this adhesion can be blocked by the appropriate synthetic oligosaccharide. For many bacteria, colonization is impossible without adhesion, and carbohydrate-based therapies for several infectious diseases, such as H. pylori-induced ulcers and pneumonia, are currently being developed.
Certain oligosaccharides are of utility for the induction of immune responses, for antibody production, as vaccines, or to induce disease states in research animals. For example, the trisaccharide nephritogenoside is used to induce glomerulonephritis in animals, and has received the attention of synthetic chemists for some time: H. Zhang, Y. Wang, W. Voelter, Tetrahedron Lett., 36, 1243-1246 (1995) and references therein.
Despite the promise of synthetic oligosaccharides for research and therapeutic applications, there is at present no universally applicable method for the synthesis of these complex molecules. Enzymatic methods have been developed recently which are quite effective for the regiospecific and stereospecific formation of glycosidic linkages, but the enzymes are fairly specific for particular substrates and not widely applicable to the variety of structures one would like to synthesize. See: O. Karthaus et al, J. Chem. Soc. Perkin Trans. 1, 1851-1857 (1994); M Schuster, P. Wang, J. Paulson, C.-H. Wong, J. Amer. Chem. Soc., 116, 1135 (1994); and references therein.
Chemical synthesis is not limited in this fashion, and in principle is capable of providing any desired oligosaccharide, and therefore carbohydrate synthesis has become a very active field of research. Novel glycosylation reactions and clever strategies for carbohydrate synthesis have been developed. Some fairly complex oligosaccharides have been synthesized, but to date there is no generally applicable "universal" method that can reliably generate glycosidic linkages with control of regiochemistry and stereochemistry. Subtle changes in the structures of glycosyl donors and acceptors change the regiochemical and stereochemical outcomes, and on the yields, of existing glycosylation reactions. Consequently, every oligosaccharide synthesis is a unique undertaking. This state of affairs does not permit the synthesis of large libraries of oligosaccharides for screening purposes. The application of combinatorial methods of synthesis requires especially reliable chemistry, and the combinatorial synthesis of oligosaccharide libraries is therefore well beyond the reach of present methods.
Thus, although the power of combinatorial synthesis for identifying drug leads and elucidating structure-activity relationships may have been appreciated for some time in specific areas of drug discovery, the combinatorial approach has not been successfully applied to the synthesis and screening of carbohydrate-based ligands, including, for example, polysaccharides and glycoconjugates. In contrast, methods to make peptides and nucleic acids on solid supports have been available for many years and so it is not surprising that the first combinatorial libraries involved peptides and nucleic acids. In contrast, methods to synthesize carbohydrates on the solid phase are only now being developed. The reliable preparation of a combinatorial oligosaccharide library requires consistently high-yielding reactions, which give well-defined products from a variety of substrates under standardized reaction conditions.
A related requirement is the availability of a set of stable carbohydrate monomers that are suitable for these reactions. These monomers should not only add to the growing oligosaccharide chain in high yield, but should carry suitable protecting groups that can be selectively removed to enable attachment of the next monomer unit. Similar requirements apply to the incorporation of carbohydrate monomer units into non-oligosaccharide libraries (e.g., glycopeptides or glycoproteins). Such a set of monomers would, of course, be useful for solid-phase or solution-phase synthesis of individual simple and complex carbohydrates, as well.
Synthesis of combinatorial libraries, especially on solid supports, puts great demands on the chemistry employed. Each step in a multi-step combinatorial library synthesis should provide the desired product with high yield, otherwise the final yield of the expected products drops to low levels. The number of by-products rises to unacceptable levels, after several rounds of synthesis. Even with yields at the level of 90% per reaction, the maximum amount of product that can be expected after only five consecutive reactions is 59%, 35% after ten such reactions. Moreover, such 90% or better yields must be obtained simultaneously, under the same reaction conditions, for the enormous number of different monomer/oligomer combinations that are present in a growing library.
There is a great need, therefore, for a method of linking saccharide monomers that can be relied on to generate regiochemically and stereochemically well defined products in high yield. Such a method would allow research workers other than skilled synthetic chemists to engage in the preparation of oligosaccharides. Likewise, there is a need for a synthetic methodology that is reliable enough to be carried out using automated equipment, analogous to the automated DNA and automated peptide synthesizers that are a mainstay of nucleic acid, protein chemistry and combinatorial library synthesis research. Both of these needs would be met by a reliable method of solid-phase oligosaccharide synthesis that approaches the kinds of results obtained using techniques currently available for peptides and nucleic acids.
2.1. Existing Methods of Oligosaccharide Synthesis
The requirements of the field of solid-phase oligosaccharide synthesis have been evident to the workers in the field of carbohydrate synthesis and much effort has, in fact, been invested in the development of various approaches with mixed results.
For instance, Schuerch et al., in Carbohydrate Res. (1972) 22:399-412, utilized glycosyl bromides as glycosyl donors to form glycosidic linkages to a polymer-supported glycosyl acceptor. However, the solid-phase reactions were much slower than the analogous solution-phase glycosylations. Consequently, the highly reactive and unstable glycosyl bromides underwent side reactions and eventual decomposition. Moreover, stereocontrol by C6 substituents, observed in solution, did not translate well to the solid-phase reactions. Thus, stereocontrol of the glycosidic linkages was also poor.
Gagnaire et al., in Tetrahedron Lett. (1972) 5065, attempted to catalyze polymer-supported glycosylation by D-glycosyl bromides with mercuric ions. They did achieve the preparation of beta(1.fwdarw.6) linkages on the solid phase by means of neighboring-group participation. They could not, however, obtain the alpha anomers cleanly unless the glycosyl acceptors were sterically hindered, and these hindered acceptors reduced the yield to about 55%.
Because of the additional difficulties presented by the poor transferability of solution-phase methods to solid-phase carbohydrate synthesis, some efforts to make carbohydrate libraries have involved the synthesis of mixtures of carbohydrates in solution.
In one attempt to eliminate the problems introduced by the solid-phase support, Guthrie et al. utilized a soluble polystyrene resin as a "solid" support together with an orthoester glycosylation method to obtain moderate yields of disaccharides (R. Guthrie, A. Jenkins, G. Roberts, J. Chem. Soc. Perkin Trans. 1, 2414 (1973)). Later, Krepinsky et al. improved on this method by utilizing a soluble polyethylene glycol polymer, and by using a glycosyl trichloroacetimidate as the glycosyl donor (S. Douglas, D. Whitfield, J. Krepinsky, J. Amer. Chem. Soc., 113, 5095 (1991)). Both Guthrie and Krepinsky attempted to retain one of the advantages of solid-phase synthesis, which is the ability to wash away spent and/or excess reagents. Both methods, therefore, require precipitation of the soluble polymer by addition of specific non-solvents. The precipitation steps must be repeated several times at each step of the synthesis, resulting in substantial material losses. In addition, the precipitation steps make automation problematic and add greatly to the time required to complete a synthesis.
A further limitation imposed by soluble "solid" supports is the fact that, when used for the synthesis of carbohydrate libraries, they do not generally permit resolution; that is, the soluble supports, even when in precipitated form, do not allow the physical separation or segregation and subsequent identification of individual members (e.g., single beads) of the library. Indeed, the library obtained from such soluble supports is a mixture at the molecular level. Therefore, in most cases, deconvolution is the only available technique for identifying library members that are of interest. There thus remains a need for a method of preparing carbohydrate libraries that are particularly amenable to resolution strategies, such as chemical tagging or spatial addressing methods or direct structure determination of product removed from the solid or polymer support.
Kahne et al. (D. Kahne, S. Walker, Y. Cheng, D. van Engen, J. Amer. Chem. Soc., 111, 6881-6882 (1989); S. Kim, D. Augeri, D. Yang, D. Kahne, J. Amer. Chem. Soc., 116, 1766-1775 (1994)) have described a sulfoxide-mediated glycosylation reaction that is high-yielding and reliable, in terms of being applicable to a variety of substrate combinations. These authors have applied this method to solid-phase synthesis of specific oligosaccharides. L. Yan, C. Taylor, R. Goodnow, D. Kahne, J. Amer. Chem. Soc., 116, 6953-6954 (1994); D. Kahne, PCT Int. Appl. Publication No. WO 94/19360 (1994). Still, where the 1,2-cis isomers of oligosaccharide products are desired, the degree of stereochemical control was found to be inadequate. Synthetic Oligosaccharides (ACS Symposium Series 560, 1994) p. 158.
Where the 1,2-trans isomers of glycosylation products are desired, it is well-known that they may be obtained selectively when a neighboring group at C-2 participates in the glycosylation. This process is illustrated in Scheme 1 (below), where a 2-.alpha.-acyloxy group (a typical participating group) on an L-glucosyl sulfoxide is shown to form a cyclic cationic intermediate, which directs the incoming acceptor to the .beta. position. As mentioned, above, where 1,2-cis isomers are desired, however, the presence of a non-participating C-2 substituent is insufficient to assure "clean" production of the 1,2-cis isomer. Consequently, unless the acceptor is sterically hindered, mixtures of products cis and trans isomers are usually obtained.
This problem of lack of stereochemical control in the case of 1,2-cis linkages has been documented by others using sulfinyl glycosides as starting substrates: Y. Wang, H. Zhang and W. Voelter obtained a 2:1 .alpha.:.beta. (cis:trans) ratio in the 6-O-glycosylation of a glucose derivative with compound III (stereochemistry=D-glucose, Ar=phenyl, all X=O, all G=benzyl) (Z. Naturforsch. 48b, 1143-1145 (1993)), and also a 2:1 ratio in the glycosylation of a disaccharide with the same donor (Z. Naturforsch. 50b, 661-666 (1995)). The same authors later reported a 4:1 .alpha.:.beta. ratio for the former reaction (Tetrahedron Lett. 36, 1243-1246 (1995)). This glycosyl donor, when equipped with a C-2 participating group (III, stereochemistry=D-glucose, Ar=phenyl, all X=O, all G=benzoyl), gave exclusively the .beta. (1,2-trans) anomer in a C-2 glycosylation reaction (T. Bamhaoud, J.-M. Lancelin, J.-M. Beau, J. Chem. Soc. Chem. Commun., 1494-1496 (1992)) and, also, in a series of 6-glycosylation reactions (L. Sliedregt, G. van der Marel, J. van Boom, Tetrahedron Lett., 35, 4015-4018 (1994)). The latter authors obtained similar results with galactose-derived donors. A similar donor III with a non-participating group at C-2 (stereochemistry=D-galactose, Ar=phenyl, all X=O, all G=benzyl) did give 1,2-cis products cleanly when glycosylating galactose derivatives at C-3 and C-4; it should be noted, however, that the starting substrates constitute examples of hindered acceptors (A. Sarkar, K. Matta, Carbohydr. Res., 233, 245-250 (1992)).
In the course of preparing the calicheamicin trisaccharide, the glycosyl donor, shown below, lacking a participating C-2 group, was reported to provide the .alpha.-anomer with 12:1 selectivity. In this particular case, however, the observed selectivity resulted from the equilibration of an initially formed product mixture. Thus, the stereoselectivity of the reaction itself is not high: S.-H. Kim, D. Augeri, D. Yang, D. Kahne, J. Amer. Chem. Soc. 116, 1766-1775 (1994). ##STR1##
The biological activity of oligosaccharides is sensitive to the stereochemistry of glycosidic linkages, and, from a biochemical, phramacologic and pharmaceutical perspective, the alpha and beta anomers of a carbohydrate are considered entirely different substances. Thus, a reaction providing a 50:50 mixture of anomers, even if it proceeds in 100% chemical yield, generates at best a 50% stereochemical yield of a single compound. As noted above, a reliable 90% or better yield at each step is minimally required for a practical multi-step library synthesis. As the number of synthetic steps rises, the required yield per step also preferably rises. The instant discussion makes it clear that no adequate methods exist in the present state of the art for the reliable construction of glycosidic linkages that present a 1,2-cis geometry, especially in the context of library synthesis, where the simultaneous .alpha.- or .beta.-glycosylation of multiple types of starting substrates is desired.
Thus, the need for reliable methods of glycosylation, which provide good chemical yields and good stereochemical control with a wide variety of substrates, remains unmet by the state of the art. In particular, there remains a need for classes of glycosyl donors that provide 1,2-cis stereochemistry in the carbohydrate product with good chemical yields and stereochemical control, especially with unhindered acceptors.